Cigarette smoking causes 443,000 premature deaths annually and $193 billion in health-related economic losses. Many are trying to quit smoking, but the majority of patients relapse to smoking within one year regardless of treatment. The long-term goal is to use molecular techniques to better characterize the genetics involved in the onset and treatment of addiction and to identify novel, individualized, treatment strategies and translate them into clinical practice. The most efficacious pharmacotherapeutic currently being used to treat tobacco dependence is varenicline, and although the mechanism through which varenicline acts on the nicotine response is theorized to act through the 42 nicotinic acetylcholine receptors, it is far from complete. Here we propose a translational zebrafish model with which we will identify key genetic factors involved in the nicotine and varenicline responses secondary to the 42 nicotinic acetylcholine receptors. The central hypothesis is genetic factors mediate the nicotine and varenicline responses involved in the treatment of nicotine dependence in humans. To test this hypothesis, the following specific aims will be used: AIM 1) Determine the impact on nicotine and varenicline response of specific genes in the nicotinic acetylcholine and GABA receptor families using knockout zebrafish. AIM 2) Identify specific genes that modulate the nicotine and varenicline responses in zebrafish using a forward genetic screen of mutants. The hypothesis, based on published and preliminary data, is nicotinic acetylcholine receptor subunit genes secondary to the 42, and the GABA receptor genes, among others, are involved in the action of nicotine and varenicline. Both nicotine and varenicline activate the nicotinic acetylcholine receptors, and upon activation of these receptors, downstream signaling causes the release of neurotransmitters such as dopamine, serotonin, glutamate and GABA. The genes involved in such secondary systems will also be critical to the nicotine and varenicline responses. For both of these aims a larval zebrafish behavioral assay followed by molecular characterization of the gene(s) and mutant(s) will be utilized. The knockout zebrafish will be obtained from the Sanger Institute Zebrafish Mutation Project, created in the laboratory using transcription activator-like effector nucleases (TALENs), or obtained elsewhere as available. The mutant zebrafish for the forward genetic screen are generated in the laboratory via a gene-breaking transposon (GBT) containing a protein trap (pGBT-RP2.1). The laboratory currently has a catalogue of 360 mutants and funding for more than 1000 additional lines that will be screened with neural lines have first priority based on the expression pattern of the mutant allele. After successful completion of these aims, the results will be valuable preliminary results for human pharmacogenetic association studies and to increase our knowledge of the underlying mechanism behind nicotine addiction and the varenicline treatment response. PUBLIC HEALTH RELEVANCE: Varenicline is the most efficacious treatment of tobacco dependence to date, but the outcomes of tobacco cessation treatment are variable and most will relapse to smoking within one year. This research will identify key genetic components involved in the response of nicotine and varenicline, which will further define how nicotine and varenicline function in the brain and will provide key insights into genes that may contribute to the variabiliy in tobacco dependence treatment response in patients. These results will lay the foundation for clinical trials looking at the genetic contribution to treatment response.